Identification and characterization of genes involved in Bacillus cereus

biofilm formation

by

J.M. Pretorius

Submitted in partial fulfillment of the requirements for the degree Master of Science

in the Faculty of Natural and Agricultural Sciences Department of Microbiology and Plant Pathology

University of Pretoria Pretoria

2010

©© UUnniivveerrssiittyy ooff PPrreettoorriiaa

DECLARATION OF ORIGINALITY

Full names of student: ..................................................................................... .

Student number: ............................................................................................ .

Declaration

1. I understand what plagiarism is and I am aware of the University's policy in this regard.

2. I declare that this thesis is my own original work. Where other people's work has been

used (either from a printed source, internet, or any other source), this has been properly

acknowledged and referenced in accordance with the departmental requirements.

3. I have not used work previously produced by another student or any other person to

hand in as my own.

4. I have not allowed, and will not allow, anyone to copy my work with the intention of

passing it off as his or her own work.

SIGNATURE STUDENT: .................................. . DATE: ...................... .

SIGNATURE SUPERVISOR: ............................. . DATE: ...................... .

ACKNOWLEDGEMENTS

Prof. V.S. BrOzel myco-supervisorwho initiatedthisproject

Dr.MarindaOosthuizenforassistancewithplanningoftheproject

Prof. A. Moir for providing us with pLTV I

The National RcscarchFoundationforfunding

Supervisor:

SUMMARY

Identification and characterization of genes involved

in Bacillus cereus biofilm formation

by

J.M. PRETORIUS

Prof.J. Theron Department of Microbiology and Plant Pathology UniversityofPretoria

Co-supervisor: Prof. V.S.BrOzel Department of Biology and Microbiology and Center for Infectious Disease Research and Vaccinology South Dakota State University

forthedegreeM.Sc

Bacillus cereus is a Gram-positive, spore-forming bacterium that is frequently identified as

the causative agent of food-borne diseases and is also implicated in food spoilage of

especially dairy products. The capacity of B. cereus to form biofilms on different substrata is

of great concern in the food industry. Not only does biofilm formation cause economic loss by

equipment failure, but contamination of food products via biofilm cells also raises safety

concerns. Bacterial biofilms have been defined as structmed multicellular communities that

form through a complex developmental process. In contrast to Gram-negative bacteria,

biofilm formation by Gram-positive bacteria has only recently been examined. Relatively few

genes have been identified that are required for these bacteria to form biofilms and little is

known about how they coordinate biofilm formation. In order to contribute to the

advancement of knowledge regarding the process of biofilm formation in Gram-positive

bacteria, the aim of this investigation was essentially to identify and characterize genes

involved in B. cereus biofilm formation.

To investigate. B. cereus ATCC 14579 was subjected to transposon mutagenesis with the

Tn917-LTVI transposon. Screening of a collection of3 500 insertional mutants for the ability

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to form biofilms at the solid-liquid-air interface of glass surfaces led to the identification of

eight biofilm-impaired mutants. Each of the mutants contained a single transposon insertion,

and no significant differences were observed in the planktonic growth rate between the B.

cereus wild-type and biofilm-impaired mutant strains. The chromosomal transposon insertion

in three of the mutants mapped to genes involved in purine biosynthesis (pur A, purC and

purL), while the transposon insertion in two other mutants mapped to the ftsE gene and to the

promoter region of the motA gene, respectively. In one of the mutants the transposon was

located in the intergenic region between two divergently transcribed genes, which encodes a

murein hydrolase exporter and nucleoside hydrolase, respectively. In the final two biofilm­

impaired mutants the transposon was respectively mapped to genes encoding a putative

membrane spanning protein and a putative protein of unknown function. Results obtained by

quantitative real-time PCR assays indicated that expression of each of the identified B. cereus

A TCC 14579 genes, with the exception of the motA gene, was up-regulated in the biofilm

population. In the case of motA, expression of the gene was down-regulated 3.2-fold in the

biofilm population and results obtained during the course of this investigation indicated that

motility, rather than the presence of flagella, is required for B. cereus biofilm formation.

Although this result is in agreement with that reported previously for B. sub til is, none of the

other genes identified in this investigation have previously been implicated in biofilm

formation by Gram-positive bacteria.

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TABLE OF CONTENTS

DECLARATION OF ORIGINALITY ............................................................. .......................... i ACKNOWLEDGEMENTS ....................................................................................................... ii SUMMARY .............................................................................................................................. iii TABLE OF CONTENTS ................................................................................. .. ........................ v LIST OF ABBREVIATIONS ................................................................................................. viii LIST OF FIGURES .................................................................................................................... X

LIST OF TABLES .......................... ......................................................................................... xii RESEARCH COMMUNICATIONS ...................................................................................... xiii CHAPTER ONE ........................................................................................................................ 1 LITERATURE REVIEW ........................................................................................................... 1

1.1 General introduction ........................................................ ................................ ........... 1 1.1.1 Bacillus cereus ................................................................................................... 1 1.1.2 Bacillus cereus ecology ...................................................................................... 2 1.1.3 Bacillus cereus pathogenesis .............................................................................. 3

1.2 Bacterial biofilms ....................................................................................................... 5 1.2.1 Biofilm formation by Bacillus species ............................................................... 6 1.2.2 Biofilm formation leads to modification of the bacterial transcriptome ............ 7 1.2.3 Biofilm formation leads to modification of the bacterial pro teo me ................... 8 1.2.4 Biofilm cells represent a distinct phenotype ...................................................... 9

1.3 Genes involved in biofilm formation by B. sub til is ................................................... 9 1.3 .1 Regulatory and associated genes ........................................................................ 9 1.3 .2 Genes involved in flagellar synthesis .... .. ............................................... .......... 10 1.3.3 Genes involved in the synthesis ofbiofilm matrix components ...................... 12 1.3.4 Other genes ....................................................................................................... 14

1.4 Quorum sensing ........................................................................................................ 1 7 1.4.1 Intraspecies communication via autoinducing peptides ................................... 18 1.4.2 Regulation of the PieR regulon via a cell-cell signalling peptide .................... 20 1.4.3 Interspecies signalling via the quorum sensing molecule AI-2 ........................ 25

1.5 Regulation ofbiofilm formation by B. subtilis ............ ............................................ 32 1.5.1 Regulation of the eps operon ................................................................. ........... 33 1.5.2 Regulation of the yqxM operon ........................................................................ 35 1.5.3 Derepression of the eps andyqxM operons by Sini ......................................... 37 1.5.4 Control of biofilm formation ............................................................................ 38

1.6 Aims of this investigation ........................................................................................ 40 CHAPTER TWO ...................................................................................................................... 42 MATERIALS AND METHODS ............................................................................................. 42

2.1 General procedures ....................................................... ...................... ...................... 42 2 .1.1 Bacterial strains ................................................................................................ 4 2 2.1.2 Plasmid and transposon .................................................................................... 42 2.1.3 Media and growth conditions ........................................................................... 44 2.1.4 Statistical analysis ......................................... ................... ................................ 44

2.2 Isolation of pL TV 1 ................................................................................................... 44 2.2.1 Plasmid extraction ............................................................................................ 44 2.2.2 Agarose gel electrophoresis ............................................................................. 45 2.2.3 Restriction enzyme digestion ........................................................................... 45 2.2.4 CsCl-EtBr density gradient centrifugation ....................................................... 45

2.3 Transformation of Bacillus cereus ATCC 14579 .................................................... 46 2.3.1 Preparation of electrocompetent cells .............................................................. 46

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2.3.2 Dialysis of plasmid DNA ................................................................................. 46 2.3.3 Electroporation ................................................................................................. 47

2.4 Construction and screening of B. cereus transposon mutant libraries ..................... 4 7 2.4.1 Transposon mutagenesis .................................................................................. 4 7 2.4.2 Identification of biofilm mutants ...................................................................... 4 7 2.4.3 Quantification ofbiofilm deficiency ................................................................ 48

2.5 Characterization of B. cereus biofilm-impaired mutants ......................................... 48 2.5.1 Growth curves ofbiofilm-impaired mutants .................................................... 48 2.5.2 Stereomicroscopy ofbiofilm-impaired mutants ............................................... 48 2.5.3 Motility assay ................................................................................................... 49 2.5.4 Transmission electron microscopy ................................................................... 49 2.5.5 Laser scanning confocal microscopy ............................................................... 49

2.6 Recovery of B. cereus chromosomal DNA flanking the transposon insertions ....... 50 2.6.1 Isolation of chromosomal DNA from biofilm-impaired mutants .................... 50 2.6.2 Preparation of competent E. coli HB 101 cells ................................................. 50 2.6.3 Cloning of the DNA flanking transposon insertions ........................................ 51 2.6.4 Isolation of plasmid DNA from E. coli HB101 ............................................... 51 2.6.5 Sequencing of flanking DNA ........................................................................... 52

2.7 Characterization of B. cereus biofilm-impaired mutants by Southern blot hybridization ......................................................................................................................... 53

2.7.1 Preparation of the labelled probe ..................................................................... 53 2.7.2 Preparation of the membrane ........................................................................... 54 2. 7.3 Hybridization .................................................................................................... 54 2.7.4 Detection of the hybridized DNA probe .......................................................... 55

2.8 Relative expression of selected B. cereus genes ...................................................... 55 2.8.1 Primers ............................................................................................................. 56 2.8.2 RNA isolation ................................................................................................... 57 2.8.3 eDNA synthesis ................................................................................................ 57 2.8.4 Reverse transcriptase (RT) negative control PCR ............................................ 58 2.8.5 Quantitative real-time PCR .............................................................................. 58

CHAPTER THREE .................................................................................................................. 60 RESULTS ................................................................................................................................. 60

3.1 Isolation of pL TV 1 ................................................................................................... 60 3.2 Transposon mutagenesis of B. cereus ATCC 14579 ................................................ 61 3.3 Screening for B. cereus biofilm-impaired mutants .................................................. 62 3.4 Characterization of B. cereus biofilm-impaired mutants by Southern blot analysis 64 3.5 Identification of disrupted genes in B. cereus biofilm-impaired mutants ................ 65 3.6 Characterization of B. cereus biofilm-impaired mutants ......................................... 68

3 .6.1 Growth curves .................................................................................................. 68 3.6.2 Microscopic analysis ofbiofilm phenotypes .................................................... 68 3.6.3 Motility assay of the biofilm-impaired mutant Mut21 ..................................... 71 3.6.4 Laser scanning confocal microscopy of the biofilm-impaired mutant Mut17. 73

3.7 Relative expression of selected genes in B. cereus biofilms .................................... 74 CHAPTER FOUR .................................................................................................................... 77 DISCUSSION .......................................................................................................................... 77

4.1 Flagella-mediated motility is required in B. cereus biofilm formation .................... 77 4.1.1 B. cereus ilmotAB is biofilm-impaired ............................................................. 77 4.1.2 The motor complex of bacterial flagella .......................................................... 77 4.1.3 B. cereus requires flagella-mediated motility for biofilm formation ............... 82

4.2 Purine biosynthesis is required in B. cereus biofilm formation ............................... 83

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4.2.1 B. cereus pur mutants are biofilm-impaired ..................................................... 83 4.2.2 B. cereus requires extracellular DNA for biofilm formation ........................... 84

4.3 A predicted ABC transporter, FtsEX, is required in B. cereus biofilm formation ... 85 4.3.1 B. cereus iJftsEX is biofilm-impaired ............................................................... 85 4.3.2 FtsEX is required for divergent activities in different bacteria ........................ 86 4.3.3 B. cereus biofilm formation is facilitated by FtsEX ......................................... 89

4.4 Different hydrolase enzymes may be required in B. cereus biofilm formation ....... 90 4.4.1 Inosine-uridine preferring nucleoside hydrolases play a role in purine salvage in protozoan parasites, and spore germination in B. thuringiensis ................................... 91 4.4.2 Murein hydro lases are involved in eDNA release and biofilm formation ....... 92 4.4.3 Additional evidence for a link between B. cereus eDNA and biofilm formation ......................................................................................................... 93

4.5 Proteins of unknown function are required in B. cereus biofilm formation ............ 95 CHAPTER FIVE .................................................................................................................. 97

CONCLUDING REMARKS ................................................................................................... 97 REFERENCES ....................................................................................................................... 101 Appendix 1 ............................................................................................................................. 121

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LIST OF ABBREVIATIONS

A Absorbance

AI-2 autoinducer-2

bp base pair

bla ~-lactamase gene

ca. approximately

cat chloramphenicol acetyltransferase gene

oc degrees Celsius

eDNA complementary DNA

cjjJ cyan fluorescent protein gene

em centimetre

CP crossing point

CsCl cesium chloride

CSF competence and sporulation factor

CTAB hexadecyltrimethylammonium bromide

d day

DEPC diethyl pyrocarbonate

dH20 distilled water

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTP deoxyribonucleoside-5 '-triphosphate

EDTA ethylenediaminetetra-acetic acid

e.g. for example

erm erythromycin resistance gene

EtOH ethanol

Fig. figure

gfp green f1 uorescent protein gene

xg centrifugal force

h hour

IPTG isopropyl ~-D-thiogalactoside

i.e. that is

kb kilobase pairs

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KOAc potassium acetate

lacZ ~-galactosidase gene

LB Luria-Bertani

litre

M molar

mg milligram

min minute

ml millilitre

mM millimolar

Mut mutant

ng nanogram

nM nanomolar

nm nanometer

nt nucleotide

ORF open reading frame

PCR polymerase chain reaction

PVC polyvinylchloride

RNA ribonucleic acid

RT reverse transcri ptase

s second

SDS sodium dodecyl sulphate

TE Tris-EDTA

2-DE two-dimensional electrophoresis

u units

j.!g microgram

j.!l micro litre

j.!M micromolar

uv ultraviolet

v. version

v volts

v/v volume per volume

w/v weight per volume

X-Gal 5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside

yfp yellow fluorescent protein gene

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LIST OF FIGURES

Figure 1.1: A general model for peptide-mediated quorum sensing in Gram-positive bacteria .......................................................................................................................................... 18

Figure 1.2: The B. sub til is ComP!ComA quorum sensing system for control of sporulation and competence ................................................................................................................ 20

Figure 1.3: Genomic region of Bacillus cereus ATCC 14579, containing the pieR and papR genes ................................................................................................................................. 23

Figure 1.4: Structure ofPapR5:PlcR and higher order structures formed in the presence of increasing concentrations of PapR5 ................................................................................. 24

Figure 1.5: AI-2-dependant quorum sensing in V. harveyi .. .................................................... 27 Figure 1.6: Biosynthesis of AI-2 from the precursor S-adenosylmethionine (SAM) .............. 28 Figure 1.7: Expression of a sspB-lacZ and a luxS-lacZ fusion by B. subtilis colonies grown on

agar containing X-Gal ...................................................................................................... 30 Figure 1.8: Genetic organization and sequence analysis of the lsr region in B. cereus A TCC

10987 and E. coli K-12 .................................................................................................... 31 Figure 1.9: Model for Lsr-mediated internalization and processing of AI-2 in E. coli . .......... 32 Figure 1.10: Effect of mutations in sinR, sin! and epsH on chain bundling, pellicle formation,

colony morphology and swarming motility ..................................................................... 34 Figure 1.11: Structure and organization of SinR and AbrB recognition sequences in the

promoter regions of the epsA-0 andyqxM-sipW-tasA operons ....................................... 36 Figure 1.12: Regulation of Bacillus multicellular behaviour ................................................... 40 Figure 2.1: Restriction map of pL TV 1 ..................................................................................... 43 Figure 2.2: Hypothetical chromosomal insertion ofTn917-LTV1 .......................................... 44 Figure 3.1: Characterization of purified pLTV1 plasmid DNA ............................................... 61 Figure 3.2: Biofilm formation by wild-type B. cereus ATCC 14579 and the derived Tn917-

L TV 1 transposon mutants ................................................................................................ 63 Figure 3.3: Southern blot analysis of chromosomal DNA extracted from B. cereus biofilm-

impaired mutants .............................................................................................................. 65 Figure 3.4: Locations of the Tn917-LTV1 insertions in the B. cereus biofilm-impaired

mutants isolated in this study ........................................................................................... 67 Figure 3.5: Growth curves of wild-type B. cereus ATCC 14579 and biofilm-impaired mutants

in LB broth at 3 7°C .......................................................................................................... 69 Figure 3.5 (continued): Growth curves of wild-type B. cereus ATCC 14579 and biofilm-

impaired mutants in LB broth at 37°C .............................................................................. 70 Figure 3.6: Phenotypes of the biofilms formed at the air-liquid interface on glass slides by

wild-type B. cereus ATCC 14579 and the biofilm-impaired mutants ............................. 71 Figure 3.7: Motility assays for wild-type B. cereus ATCC 14579 (WT) and the motAB mutant

strain Mut21 ..................................................................................................................... 72 Figure 3.8: Representative transmission electron micrographs of negatively-stained cells from

wild-type B. cereus ATCC 14579 (WT) and the motAB mutant strain Mut21 ................ 73 Figure 3.9: Representative laser scanning confocal micrographs of planktonic cells from wild-

type B. cereus ATCC 14579 and theflsEX mutant strain Mut17 .................................... 74 Figure 3.10: Whisker-box plot indicating the ratio of gene transcripts in a biofilm to the gene

transcripts in the planktonic population quantified by quadruplicate real-time PCR reactions using the 16S rRNA gene as endogenous control.. ........................................... 76

Figure 4.1: Schematic representation of the structure and components of the Salmonella enterica serovar Typhimurium flagellum ........................................................................ 78

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Figure 4.2: Alignment of amino acid sequences of the B. cereus ATCC 14579 stator components with the amino acid sequence of MotA, MotB, MotP and MotS of B. sub til is .......................................................................................................................................... 81

Figure 4.3: Transcriptional organization of the motAB operon of B. cereus ATCC 14579 ..... 82 Figure 4.4: ClustalW2 alignment of amino acid sequences of B. cereus ATCC 14579 BC5133

and S. aureus MW2 CidA ................................................................................................ 95

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LIST OF TABLES

Table 2.1: Real-time PCR primers used for relative quantification of gene expression in biofilm versus planktonic cells ......................................................................................... 56

Table 3.1: Descriptive statistics of real-time PCR reactions .................................................... 76

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RESEARCH COMMUNICATIONS

Papers published:

1. Vilain, S., Pretorius, J.M., Theron, J. and Brozel, V.S. (2009). DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilm. Applied and Environmental Microbiology 75, 2861-2868.

Conference contributions:

1. Pretorius, J.M., Brozel, V.S. and Theron, J. (2003). Identification of genes involved in Bacillus cereus biofilm formation. 18th Congress of the South African Society of Biochemistry and Molecular Biology (SASBMB), 6-9 July 2003, Pretoria, South Africa.

2. Pretorius, J.M., Brozel, V.S. and Theron, J. (2004). Identification of genes involved in Bacillus cereus biofilm formation. Joint Congress of the South African Microbiology Society and South African Genetics Society, 4-7 April 2004, Stellenbosch, South Africa.

3. Pretorius, J.M., Brozel, V.S. and Theron, J. (2006). Characterisation of Bacillus cereus biofilm-impaired transposon mutants. 14th Biennial Congress of the South African Society for Microbiology, 9-12 April2006, Pretoria, South Africa.

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